Warsaw Agricultural University, Warsaw, Poland PARASITISM OF RUST FUNGI SPORES BY RAMULARIA SPECIES A. Bartkowska Abstract Three Ramularia species have been known as hyperparasites of rust fungi in Poland. Ramularia coleosporii is quite frequently found on uredinial sori of Coleosporium tussilaginis; R. uredinis parasitizes uredinial sori of many species belonging to Melampsora and Cronartium genera, whereas R. uredinearum is a parasite of various sori of Puccinia species. Uredinial and telial sori of different rust fungi species infected with three Ramularia species were examined using light, scanning and transmission microscopes to determine the parasite-hyperparasite contact. The examination clearly showed that mycelium of Ramularia species grows on the surface of the uredinial and telial sori of rust fungi. Hyphae of the hyperparasite directly penetrate rust spores causing gradually their complete destruction. Key words: Ramularia coleosporii, Ramularia uredinis, Ramularia uredinearum, hyperparasitism Introduction Ramularia coleosporii, R. uredinis and R. uredinearum are quite common parasites of uredinial and telial stage of a few species of rust fungi. The hyperparasites are most commonly observed as white and usually dense mycelium produced in uredinial and telial sori as well. Only information concerning morphology of hyperparasite can be found in foreign literature (Kirulis 1942, Braun 1998). Ramularia coleosporii has been known from a few European, Asian and American countries as a parasite of rust pustules from Coleosporium and Chrysomyxa genus (Kirulis 1942, Braun 1998). It has been also often collected from Poland as a parasite of Coleosporium genus only (Dominik 1936, Kućmierz 1967, 1971, 1976, Mułenko 1988 (1989), Danilkiewicz 1990, Wołczańska 2005). So far, R. uredinearum Phytopathol. Pol. 43: 61 67 The Polish Phytopathological Society, Poznań 2007 ISSN 1230-0462
62 A. Bartkowska has not been studied sufficiently due to its rare occurrence. For the first time it was described from aecia of Puccinia cerithes-agropyrina Tranzsch. (Aecidium asperifolii Pers.) from Romania, afterwards it was collected from Taiwan, European countries (Braun 1998) and from Poland (Dominik 1936, Majewski 1970) on different types of Pucciniaceae family rust pustules. The third species, R. uredinis has been known as a hyperparasite of rust fungi from Cronartium, Melampsora, Melampsoridium, Pucciniastrum and Phragmidium genus. It was collected from a few Asian and European countries (Braun 1998) and also from Poland (Mułenko 1988 (1989), Wołczańska 2005). This study reports on morphology of the rust fungi and three Ramularia species host-parasite interface, showing the nutrient structures and the effect of parasitism on the fungal host. Materials and methods Samples of Tussilago farfara with Coleosporium tussilaginis and R. coleosporii, Rubus plicatus with Phragmidium sp. and R. uredinis as well as Aegopodium podagraria with Puccinia aegopodii and R. uredinearum were collected from the Warsaw Agricultural University area and Kampinos Forest. Dry leaf pieces bearing rust pustules overgrown by the fungi (R. coleosporii, R. uredinearum and R. uredinis) were cut into 5 5 mm pieces and then coated with carbon and silver in an JEOL JEE-4C vacuum evaporator and finally examined with a JEOL JSM-35 scanning electron microscope operating at 25 kv. For transmission electron microscopy infected leaves were cut into small fragments (3 3 mm) and fixed for 2 h in 4% paraformaldehyde and 5% glutaraldehyde in 0.2 M cacodylic buffer (ph 7.0) at 4 C. After several rinses in buffer, the samples were post-fixed with 2% OsO 4 in the same buffer for 2 h. Samples were dehydrated in a graded series of ethanol and embedded in Epon 812. Polymerization lasted 24 h at 60 C. Sections were cut on a Ultracut E (Reichert) microtome. Ultrathin sections were mounted on copper grids, stained for 20 min in uranyl acetate and Reynold s citrate for 30 min and finally examined in a JEOL JEM 1220 in the Laboratory of Electron Microscopy of the Warsaw Agricultural University. Results Three species of hyperparasites: R. coleosporii, R. uredinis and R. uredinearum grew on the surface of uredinial and telial sori and between spores of rust fungi. The mycelium of R. uredinearum is usually invisible on the surface of rust sori. The only visible structures are conidiophores with conidia outgrowing among host spores (Phot. 1). However, the mycelium consisting of dense hyphae net and conidiophores with spores of R. uredinis and R. coleosporii grow extensively on the surface and inside uredinia and telia (Phot. 2). The hyphae of R. coleosporii grow visibly
Parasitism of rust fungi spores by Ramularia species 63 Phot. 1. Conidiophores of Ramularia uredinearum growing out of a telium of Puccinia aegopodii (photo by A. Bartkowska) Phot. 2. The extensive development of Ramularia uredinis mycelium within an uredinium of Phragmidium sp. (photo by A. Bartkowska)
64 A. Bartkowska Phot. 3. Direct growth of the Ramularia coleosporii hyphae towards an urediniospore of Coleosporium tussilaginis (photo by A. Bartkowska) Phot. 4. A callosity similar to appressorium in the end of an infection hypha of Ramularia uredinis adhesing to the surface of a Phragmidium sp. spore wall (photo by A. Bartkowska)
Parasitism of rust fungi spores by Ramularia species 65 Phot. 5. The hyperparasite (Ramularia uredinis) sporulation on remnants of an urediniospore (Phragmidium sp.) (photo by A. Bartkowska) Phot. 6. Degradation of the content of a Phragmidium sp. urediniospore (circle) in contact with Ramularia uredinis hyphae (arrows) (scale bar: 2 m) (photo by A. Bartkowska)
66 A. Bartkowska towards the host urediniospores (Phot. 3). Moreover, little callosities similar to appressoria were observed in the ends of the infection hyphae adhesing to the surface of the spore walls (Phot. 4). Nevertheless, haustoria were not found in the cross section of infected spores, therefore the decaying host spores are penetrated with hyphae of hyperparasite. In consequence of Ramularia sp. strong infection, walls and contents of rust spores are subjected to complete destruction (Phot. 6). It is very frequent when hyperparasite forms conidiophores on the surface of the infected rust spores (Phot. 5). Discussion Till now there are few reports concerning the occurrence and morphological description of the three Ramularia species on rust fungi (Dominik 1936, Kirulis 1942, Kućmierz 1967, 1971, 1976, Majewski 1970, Mułenko 1988 (1989), Braun 1998, Danilkiewicz 1990, Wołczańska 2005). However, there are no results reported on parasitism of rusts by Ramularia species to be compared with the above mentioned data. The results are a reference point to be considered in further studies on Ramularia species parasitizing rust fungi. In this study R. coleosporii, R. uredinis and R. uredinearum were for the first time confirmed as a hyperparasites of rust fungi spores. The results indicate that the Ramularia spp. studied attacked urediniospores and teliospores. Hyperparasites penetrated the host spores directly and caused their rapid destruction. This rapid killing may result from the action of enzymes secreted by hyperparasites. This mode of parasitism is, however, not understood sufficiently and needs further studies in the future. Streszczenie GRZYBY RODZAJU RAMULARIA JAKO PASOŻYTY ZARODNIKÓW GRZYBÓW RDZAWNIKOWYCH Wykazano kontakt grzybni trzech gatunków nadpasożytów rodzaju Ramularia z kilkoma gatunkami grzybów rdzawnikowych. Udokumentowano rozwój grzybni na urediniach i teliach, kierowanie się strzępek infekcyjnych ku zarodnikom oraz następującą destrukcję porażonych urediniospor. Stwierdzono, że strzępki infekujące tworzą niekiedy na końcu zgrubienie przypominające appressorium. Literature Braun U., 1998: A monograph of Cercosporella, Ramularia and allied genera (Phytopathogenic Hyphomycetes). Vol. 2. IHW, Eching.
Parasitism of rust fungi spores by Ramularia species 67 Danilkiewicz M., 1990: Grzyby pasożytnicze lewobrzeżnej doliny środkowego Bugu. Acta Mycol. 23, 2: 37 80. Dominik T., 1936: Materjały do flory grzybów mikroskopowych zachodniej Polski. Spraw. Kom. Fizjogr. PAU 70: 1 72. Kirulis A., 1942: Mikroskopikâssçnes kâ augu slimîbu dabîgie ienaidnieki Latvijâ [Die mikroskopischen Pilze als natürliche Feinde der Pflanzenkrankheiten in Lettland]. Jelgavas Lauksaimniecîbas Akad. Raksti, Lauksaimniecîbas Fak. 1: 478 536. Kućmierz J., 1967: Materiały do flory grzybów pasożytniczych Polski. Rocz. Nauk.-Dydakt. WSP Krak. Pr. Bot. 28: 93 107. Kućmierz J., 1971: Grzyby pasożytnicze Ojcowskiego Parku Narodowego. Część III. Workowce (Ascomycetes), grzyby niedoskonałe (Deuteromycetes). Fragm. Florist. Geobot. 17: 425 438. Kućmierz J., 1976: Flora grzybów pasożytniczych Pienin. Część II. Basidiomycetes, Deuteromycetes. Fragm. Florist. Geobot. 22: 605 622. Majewski T., 1970: Peronosporales, Erysiphaceae, Uredinales, Ustilaginales. In: Mikoflora Bieszczadów Zachodnich. IV. (Zatwarnica, 1965). Eds. S. Domański, M. Lisiewska, T. Majewski, A. Skirgiełło, W. Truszkowska, W. Wojewoda. Acta Mycol. 6, 1: 135 139, 165 173. Mułenko W., 1988 (1989): Mikroskopowe grzyby fitopatogeniczne Pojezierza Łęczyńsko-Włodawskiego. II. Acta Mycol. 24: 125 171. Wołczańska A., 2005: Grzyby z rodzaju Ramularia występujące w Polsce. Monogr. Bot. 95. Author s address: Dr. Agnieszka Bartkowska, Warsaw Agricultural University, Department of Plant Pathology, ul. Nowoursynowska 159, 02-776 Warsaw, Poland, e-mail: agnieszka_plachecka@sggw.pl Accepted for publication: 31.03.2007